Formation of collagen fibrils in vitro by cleavage of procollagen with procollagen proteinases.
A new system was developed for studying the assembly of collagen fibrils in vitro. A partially purified enzyme preparation containing both procollagen N-proteinase and c-proteinase (EC 3.4.24.00) activities was used to initiate fibril formation by removal of the N- and C-propeptides from type I procollagen in a physiological buffer at 35-37 degrees C. The kinetics of fibril formation were similar to those observed for fibril formation with tissue-extracted collagen in the same buffer system, except that the lag phase was longer. The longer lag phase was in part accounted for by the time required to convert procollagen to collagen. Similar results were obtained when an intermediate containing the C-propeptide but not the N-propeptide was used as a substrate. Therefore, removal of the c-propeptide appeared to be the critical step for fibril formation under the conditions used here. The fibrils formed by enzymic cleavage of procollagen or pCcollagen appeared microscopically to be more tightly packed than fibrils formed directly from collagen under the same conditions. This impression was confirmed by the observation that the fibrils formed by cleavage of procollagen were stable to temperatures 1.5-2 degrees C higher than fibers formed from extracted collagen under the same conditions. When smaller amounts of procollagen proteinase were used, the rate of cleavage of procollagen to collagen was markedly reduced. The fibrils which formed under these conditions were up to 3 micrometers in diameter. Some appeared to contain branch points.
D000818:Animals; D055395:Bone Morphogenetic Protein 1; D019485:Bone Morphogenetic Proteins; D001842:Bone and Bones / Q000201:enzymology; D002642:Chick Embryo; D003094:Collagen / Q000096:biosynthesis*; D004355:Drug Stability; D010450:Endopeptidases / Q000378:metabolism*; D006358:Hot Temperature; D007700:Kinetics; D008666:Metalloendopeptidases; D008854:Microscopy, Electron; D011347:Procollagen / Q000378:metabolism*; D011348:Procollagen N-Endopeptidase; D011489:Protein Denaturation
D019485:Bone Morphogenetic Proteins; D011347:Procollagen; D003094:Collagen; D010450:Endopeptidases; D008666:Metalloendopeptidases; D011348:Procollagen N-Endopeptidase; D055395:Bone Morphogenetic Protein 1